Cloning systems for Rhodococcus and related bacteria

Abstract

A plasmid transformation system for Rhodococcus was developed using an Escherichia coli-Rhodococcus shuttle plasmid. Rhodococcus sp. H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200 and pMVS300, of 75, 19.5 and 13.4 kilobases (Kb), respectively. A 3.8 Kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3 Kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla) as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1 Kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1 (pMVS301) and transformed into Rhodococcus sp. AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. This strain was deposited with the ATCC on Feb. 1, 1988 and assigned ATCC 53719. The plasmid contains the Rhodococcus origin of replication. The plasmid and derivatives thereof can therefore be used to introduce nucleic acid sequences to and from Rhodococcus for subsequent expression and translation into protein. The isolated origin of replication can also be used in the construction of new vectors.

We claim: 
     
       1. A cloning vector comprising: an origin of replication as present in plasmid pMVS301 in Rhodococcus AS50-1 (pMVS100, pMVS200, pMVS301) ATCC 53719 deposited Feb. 1, 1988.   
     
     
       2. The cloning vector of claim 1 wherein the origin of replication is in a plasmid. 
     
     
       3. The cloning vector of claim 2 wherein the plasmid is selected from the group consisting of pMVS301, (ATCC 53719) pMVS302, pMVS301KC, and pMVS301KA. 
     
     
       4. The cloning vector of claim 2 wherein the plasmid contains nucleic acid sequences in addition to the sequences contained in pMVS301 (ATCC 53719). 
     
     
       5. The cloning vector of claim 2 further comprising a nucleic acid sequence encoding a protein. 
     
     
       6. The cloning vector of claim 3 further comprising an origin of replication for a bacteria in a genus other than Rhodococcus. 
     
     
       7. The cloning vector of claim 6 wherein the non-Rhodococcus origin of replication is an E. coli origin of replication. 
     
     
       8. A host selected from the group consisting of Rhodococcus sp., Actinomyces, Nocardia, Mycobateria, and Streptomyces haboring the vector of claim 1. 
     
     
       9. The cloning vector of claim 8 wherein the host is Rhodococcus AS50-1 (pMVS100, pMVS200, pMVS301) (ATCC 53719). 
     
     
       10. A method for cloning and expressing nucleic acid sequences in Rhodococcus and related bacteria capable of using the same origin of replication comprising: providing a vector capable of stable replication in   Rhodococcus containing an origin of replication for use in Rhodococcus present in Rhodococcus AS50-1 (pMVS100, pMVS200, pMVS301) (ATCC 53719);   transforming a host bacteria capable of recognizing the Rhodococcus origin of replication; and   culturing the transformed host bacteria under conditions wherein nucleic acid sequences are expressed.   
     
     
       11. The method of claim 10 wherein the vector is a plasmid. 
     
     
       12. The method of claim 11 wherein the plasmid is selected from the group consisting of pMVS301, (ATCC 53719) pMVS302, pMVS301KC, pMVS301KA and derivatives of these plasmids containing the pMVS301 origin of replication. 
     
     
       13. The method of claim 11 further comprising: providing a sequence encoding a gene and inserting the sequence into the plasmid.   
     
     
       14. The method of claim 10 further comprising providing a host selected from the group consisting of Rhodococcus sp., Actinomyces, Nocardia, Mycobacteria, and Streptomyces. 
     
     
       15. The method of claim 14 wherein the host is selected from the group consisting of Rhodococcus AS50-1 (pMVS301) ATCC 53719 deposited Feb. 1, 1988 and derivatives thereof, R. equi, R. erythropolis, and R. globulerus. 
     
     
       16. The method of claim 11 further comprising providing a gene for antibiotic resistance in the plasmid. 
     
     
       17. The method of claim 10 wherein the origin of replication is selected from the origins of replication in plasmids pMVS100 and pMVS200.